Lipid-based nanoparticles and methods using same

ABSTRACT

The invention provides an improved lipid-based nanoparticle, which can be used to deliver a therapeutic agent to a subject, such as but not limited to a mammal, such as but not limited to a human. In certain embodiments, the nanoparticle of the invention has reduced aggregation properties as compared to those taught in the prior art.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, PCTInternational Application No. PCT/US2018/022182, filed Mar. 13, 2018,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application No. 62/470,478, filed Mar. 13, 2017, all of whichapplications are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Phospholipid nanoparticles of diameter lower than about 100 nm are oftenused as carriers to improve in vivo delivery of active pharmaceuticalingredients (APIs), such as peptides and biogenic amines. Thenanoparticles' small particle size (which is comparable to those ofsmall viruses) allows them to easily cross membrane barriers. Further,nanoparticles may provide rapid and specific delivery of APIs to desiredcell surface receptors, resulting in improved pharmacological action andneed for lower API doses. The targeted API delivery also leads to lowertoxicity, because of the API's reduced delivery to unwanted tissues inthe body.

An example of such nanoparticles is the hepatic delivery vesicle (HDV),which comprises a hepatocyte-targeting component and delivers APIs tohepatic receptors. In contrast, nanoparticles without ahepatocyte-targeting components generally accumulate in livermacrophages called Kupffer cells, along with other macrophage cells inthe body.

Diabetes mellitus, encompassing Type I and Type II forms, is a disorderaffecting large numbers of people worldwide. Diabetes mellitusmanagement comprises normalizing blood glucose levels in the subject,and that may require multiple daily injections of an insulin-basedproduct. Despite the presence of various insulin-based products on themarket, there is still a need for novel insulin-containing formulationsthat control glucose blood levels in the subject over a wide period oftime.

Most medications approved for diabetes mellitus treatment comprise aninsulin analog that is to be administered subcutaneously, often as atime-release formulation. Such administration releases the insulinanalog to peripheral tissues, but generally not to the liver. In oneaspect, proper diabetes mellitus treatment requires an insulin-basedformulation in which a 35849741.1 portion of the dosed insulin isreleased to peripheral tissues throughout the day and another portion ofthe dosed insulin is targeted for liver delivery. Such need extends aswell to other therapeutic agents for which targeted liver delivery hasadvantageous pharmacological and/or therapeutic properties.

There is thus an unmet need in the art for compositions and methods foradministering a therapeutic agent to a subject, such that thetherapeutic agent is delivered to peripheral tissues as well as to theliver of the subject. Such therapeutic agents comprise, in anon-limiting example, insulin or any analog thereof, which can be usedto manage blood glucose levels in Type I and Type II diabetic patients.The present invention meets this need.

BRIEF SUMMARY OF THE INVENTION

The invention provides a composition comprising a lipid-basednanoparticle. The invention further comprises a method of preparing thelipid-based nanoparticle of the invention. The invention furtherprovides a method of treating a disease in a mammal. The inventionfurther provides a method of activating hepatic glycogen synthase in amammal.

In certain embodiments, the nanoparticle is enclosed by a bipolar lipidmembrane. In other embodiments, the membrane comprises cholesterol,dicetyl phosphate, an amphipathic lipid and a hepatocyte receptorbinding molecule. In yet other embodiments, the amphipathic lipidcomprises at least one selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In yet other embodiments,the membrane comprises at least one agent selected from the groupconsisting of a stabilizer and stearoyl lysophosphatidylcholine. In yetother embodiments, the stabilizer is selected from the group consistingof m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate, thiomersal, andbutylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol). In yetother embodiments, the stabilizer ranges from about 10% to about 25%(w/w) in the membrane. In yet other embodiments, the stearoyllysophosphatidylcholine ranges from about 5% to about 30% (w/w) in themembrane. In yet other embodiments, the at least one hepatocyte receptorbinding molecule extends outward from the nanoparticle. In yet otherembodiments, the size of the nanoparticle ranges from about 10 nm toabout 150 nm.

In certain embodiments, the nanoparticle is enclosed by a bipolar lipidmembrane. In other embodiments, the membrane comprises cholesterol,dicetyl phosphate, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),and 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate (biotin DHPE). In yet other embodiments, the membranefurther comprises at least one agent selected from the group consistingof stearoyl lysophosphatidylcholine and m-cresol. In yet otherembodiments, the membrane comprises cholesterol, dicetyl phosphate,DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotin DHPE in a %(w/w) ratio selected from the group consisting of: (a) about9.4:18.1:56.8:14.1:0.0:1.5; (b) about 7.7:15.0:58.6:0.0:17.4:1.3; and(c) about 8.4:16.2:47.5:7.6:19.0:1.3. In yet other embodiments, thebiotin-DHPE extends outward from the nanoparticle. In yet otherembodiments, the size of the nanoparticle ranges from about 10 nm toabout 150 nm.

In certain embodiments, a therapeutic agent is dispersed within thenanoparticle. In other embodiments, the therapeutic agent is covalentlybound to the nanoparticle. In yet other embodiments, the therapeuticagent is not covalently bound to the nanoparticle. In yet otherembodiments, the nanoparticle is suspended in an aqueous solutioncomprising a free dissolved therapeutic agent that is not dispersedwithin the nanoparticle. In yet other embodiments, the therapeutic agentcomprises at least one selected from the group consisting of insulin,insulin analogs, interferon, parathyroid hormone, calcitonin, serotonin,serotonin agonist, serotonin reuptake inhibitor, human growth hormone,GIP, anti-GIP monoclonal antibody, metformin, bromocriptine, dopamine,glucagon, amylin, and GLP-1. In yet other embodiments, the therapeuticagent is insulin. In yet other embodiments, the nanoparticle-dispersedinsulin and the free dissolved insulin are independently selected fromthe group consisting of insulin lispro, insulin aspart, regular insulin,insulin glargine, insulin zinc, extended human insulin zinc suspension,isophane insulin, human buffered regular insulin, insulin glulisine,recombinant human regular insulin, recombinant human insulin isophane,and any combinations thereof.

In certain embodiments, the amphipathic lipid comprises at least oneselected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

In certain embodiments, the hepatocyte receptor binding moleculecomprises biotin. In certain embodiments, the biotin-containinghepatocyte receptor binding molecule comprises at least one selectedfrom the group consisting of N-hydroxysuccinimide (NHS) biotin;sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; ρ-aminobenzoylbiocytin trifluoroacetate; ρ-diazobenzoyl biocytin; biotin DHPE(2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate); biotin-X-DHPE (2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; and biotin6-O-phospho-α-D-mannopyranoside. In other embodiments, thebiotin-containing hepatocyte receptor binding molecule is biotin DHPE.In yet other embodiments, the biotin-containing hepatocyte receptorbinding molecule is biotin-X-DHPE. In yet other embodiments, thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE.

In certain embodiments, the composition further comprises celluloseacetate phthalate, which is at least partially bound to the therapeuticagent dispersed within the nanoparticle.

In certain embodiments, the composition further comprises at least onecharged organic molecule associated with the therapeutic agent dispersedwithin the nanoparticle, wherein the charged organic molecule is atleast one selected from the group consisting of protamines, polylysine,poly (arg-pro-thr)_(n) in a mole ratio of 1:1:1, poly(DL-Ala-poly-L-lys)_(n) in a mole ratio of 6:1, histones, sugar polymerscomprising a primary amino group, polynucleotides with primary aminogroups, proteins comprising amino acid residues with carboxyl (COO⁻) orsulfhydral (S⁻) functional groups, and acidic polymers.

In certain embodiments, the cholesterol ranges from about 5% to about15% (w/w) in the membrane.

In certain embodiments, the dicetyl phosphate ranges from about 10% toabout 25% (w/w) in the membrane.

In certain embodiments, the DSPC ranges from about 40% to about 75%(w/w) in the membrane.

In certain embodiments, the hepatocyte receptor binding molecule rangesfrom about 0.5% to about 4% (w/w) in the membrane.

In certain embodiments, the amount of the stearoyllysophosphatidylcholine in the membrane is about 5%-30% (w/w) of theamount of DSPC in the membrane.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, m-cresol, and at least one selected from the groupconsisting of biotin DHPE and biotin-X-DHPE.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and at least oneselected from the group consisting of biotin DHPE and biotin-X-DHPE.

In certain embodiments, the method comprises contacting in an aqueoussystem cholesterol, dicetyl phosphate, amphipathic lipid, hepatocytereceptor binding molecule, and the at least one agent. In otherembodiments, the method comprises contacting in an aqueous systemcholesterol, dicetyl phosphate, DSPC, biotin-DHPE, and the at least oneagent. In yet other embodiments, the at least one agent comprises astabilizer, which is added to the aqueous system after the cholesterol,dicetyl phosphate, amphipathic lipid, stearoyl lysophosphatidylcholineif present, and hepatocyte receptor binding molecule had been contactedin the aqueous system. In yet other embodiments, the at least one agentis m-cresol and is added to the aqueous system after the cholesterol,dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine if present,and biotin-DHPE had been contacted in the aqueous system. In yet otherembodiments, the nanoparticle comprises a therapeutic agent dispersedtherewithin. In yet other embodiments, the therapeutic agent,cholesterol, dicetyl phosphate, amphipathic lipid, hepatocyte receptorbinding molecule, and the at least one agent are simultaneouslycontacted in the aqueous system. In yet other embodiments, thetherapeutic agent, cholesterol, dicetyl phosphate, DSPC, at least oneagent, and biotin-DHPE are simultaneously contacted in the aqueoussystem. In yet other embodiments, the nanoparticle is formed in theabsence of the therapeutic agent, wherein optionally the nanoparticle isat least partially concentrated, purified or isolated, and wherein thetherapeutic agent is contacted with the nanoparticle, whereby at least aportion of the therapeutic agent is dispersed within the nanoparticle.In yet other embodiments, the nanoparticle comprises a therapeutic agentdispersed therewithin.

In certain embodiments, the method comprises administering to the mammalin need thereof a therapeutically effective amount of a composition ofthe invention. In other embodiments, the disease is diabetes mellitusand the therapeutic agent comprises insulin.

In certain embodiments, the method comprises administering to the mammalin need thereof a therapeutically effective amount of a composition ofthe invention, wherein the therapeutic agent comprises insulin. In otherembodiments, the mammal has diabetes mellitus.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purposes of illustrating the invention, there are depicted inthe drawings certain embodiments of the invention. However, theinvention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings.

FIGS. 1A-1B illustrate an image of a glass vial comprising non-limitinglipid-based nanoparticles of the invention. FIG. 1A: The vast majorityof nanoparticles are not visible in the image due to their small size(<100 nm). The visible nanoparticle aggregates, which can be disrupted,are about 1-3 mm. FIG. 1B is an enlargement of a selected section ofFIG. 1A.

FIG. 2 comprises a graph illustrating selected results from an insulindeficient dog oral glucose tolerance test. Formulations A (-▴-) and B(-•-) were compared to control Lispro-insulin (-▪-) (0.125 U/kg).

FIG. 3 comprises a graph illustrating various ingredient concentrations(in mg/mL) in compositions of the invention, as a function of %lysolecithin (or lysophosphatidylcholine) in the initial composition.The graph illustrates effect of initial lysolecithin concentration oncomposition stability.

FIG. 4 comprises a graph illustrating reduction of lysolecithin formed(in mg/mL bulk HDV) as a function of % lysolecithin of total lecithinconcentration. The graph illustrates effect of initial lysolecithinconcentration in production of lysolecithin over time.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates in part to an improved lipid-based nanoparticle,which can be used to deliver a therapeutic agent to a subject, such asbut not limited to a mammal, such as but not limited to a human. Incertain embodiments, the nanoparticle of the invention has reduced orminimal aggregation properties as compared to those taught in the priorart, such as but not limited to those recited in U.S. Patent ApplicationNos. US20110135725 and US20090087479, all of which are incorporatedherein in their entireties by reference. In other embodiments, thereduced or minimal aggregation properties of the nanoparticle of theinvention improves its stability and pharmaceutical developability ascompared to nanoparticles of the prior art.

In certain embodiments, the lipid-based nanoparticle of the invention isdefined and/or enclosed by a bipolar lipid membrane. In otherembodiments, the nanoparticle of the invention comprises ahepatocyte-targeting compound, which helps deliver the therapeutic agentassociated with, and/or dispersed within, the nanoparticle to ahepatocyte. In yet other embodiments, the nanoparticle of the inventionis part of a composition further comprising a “free” therapeutic agent,which is not associated with, and/or dispersed within, the nanoparticle.The nanoparticle, and any compositions comprising the same, can beadministered by any compatible and/or feasible routes, such as but notlimited to by injection (such as, for example, subcutaneously and/ortransdermally), inhalationally, buccally and/or orally, so as to treat asubject that benefits from administration of the therapeutic agentassociated with, and/or dispersed within, the nanoparticle, and/or ofthe “free” therapeutic agent, which is not associated with, and/ordispersed within, the nanoparticle.

Liposomes usually comprise amphipathic phospholipid materials that formbilayer membranes that define and/or enclose the liposomes. They canhave a single membrane (unilamellar), or multiple bilayers with amicroscopic onion-like appearance. Liposomes can be rather large,measuring several microns in diameter. Liposomes generally have aspherical (or nearly spherical) shape, wherein the intact surface has noavailable “open” edges and thus cannot interact with other available“open” edge liposome(s) to undergo particle aggregation. In contrast,phospholipid nanoparticles with diameters equal to or lower than about200 nm have a restricted ability to bend into a spherical configuration,which should in principle be their thermodynamically stable structure.As a result, these low-diameter nanoparticles do not form a perfectlyspherical particle, but rather a nearly planar sheet. Without wishing tobe limited by any theory, those nearly planar sheets can be described as“nanodiscs” or “nanoFrisbees” or “bicelles.” Such nanoparticles have“open” edges in their membranes, and these “edges” act as sticky pointsthat can promote nanoparticle aggregation. As a result, in manyinstances the nanoparticles are generated as discrete particles, whichthan proceed to aggregate into larger, easily visible (wispy orfeather-like) floating particles. This phenomenon may hamper thedevelopability of the low-diameter nanoparticles as drug deliveryagents. In certain embodiments, unlike in the case of liposomes, the APIis not carried in the core volume of (or within) the bicelles. In otherembodiments, the API is attached and/or bound to the membrane surface ofthe bicelles, either through a purely physical interaction or a covalentlinkage. In one aspect, the present invention addresses this issue,providing compositions and methods that allow for closing the “open”edges of the nearly planar sheets (nanodiscs and/or nanoFrisbees) andthus minimizing or suppressing their tendency to self-aggregate.

As described herein, the lipid-based nanoparticles of the invention areuseful as pharmaceutical carriers, and do not form the wispy,feathery-like structures described elsewhere herein. In certainembodiments, the nanoparticles of the invention comprise certainamphipathic lipids and/or certain organic molecules that enable the“open” edges of the planar nanoparticle membranes to be changed in a waythat prevents aggregation of the nanoparticles.

In certain embodiments, appropriate closing of the “open” edges of thelipid-based nanoparticle is promoted by replacing a portion ofdistearoyl phosphatidylcholine [also known as(S)-2,3-bis(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate orDSPC, which comprises two C₁₈ acyl groups covalently linked to aglycerol backbone] with a C₁₂-C₂₄ acyl lysophosphatidylcholine [alsoknown as C₁₂-C₂₄ acyl lysolecithin, or 1-(C₁₂-C₂₄acyl)-sn-glycero-3-phosphocholine, or (S)-2-hydroxy-3-(C₁₂-C₂₄acyloxy)propyl (2-(trimethylammonio)ethyl) phosphate, which comprises asingle C₁₂-C₂₄ acyl group covalently linked to a glycerol backbone]:

In certain embodiments, appropriate closing of the “open” edges of thelipid-based nanoparticle is promoted by replacing a portion ofdistearoyl phosphatidylcholine [also known as(S)-2,3-bis(stearoyloxy)propyl (2-(trimethylammonio)ethyl) phosphate orDSPC, which comprises two C₁₈ acyl groups covalently linked to aglycerol backbone] with stearoyl lysophosphatidylcholine [also known as1-steroyl-sn-glycero-3-phosphocholine, or(S)-2-hydroxy-3-(stearoyloxy)propyl (2-(trimethylammonio)ethyl)phosphate, which comprises a single C₁₈ acyl group covalently linked toa glycerol backbone]:

In certain embodiments, when incorporated into the membrane, a C₁₂-C₂₄acyl lysophosphatidylcholine (such as but not limited to stearoyllysophosphatidylcholine) prevents and/or minimizes the aggregation thatoccurs when that compound is omitted from the membrane. In otherembodiments, the C₁₂-C₂₄ acyl lysophosphatidylcholine (such as but notlimited to stearoyl lysophosphatidylcholine), with its single aliphaticchain, enables closure of any existing membrane “edge” in thenanoparticle.

In certain embodiments, when incorporated into the membrane, any ofcertain small molecule stabilizers or any salts and/or solvates thereof,such as but not limited to m-cresol, benzyl alcohol, methyl4-hydroxybenzoate, thiomersal, and butylated hydroxytoluene (also knownas 2,6-di-tert-butyl-4-methylphenol), prevents and/or minimizes theaggregation that occurs when that compound is omitted from the membrane.In other embodiments, the small molecule stabilizers or any salts and/orsolvates thereof enable closure of any existing membrane “edges” in thenanoparticle.

In certain embodiments, when incorporated into the membrane, anycombinations of any of certain small molecule stabilizers or any saltsand/or solvates thereof, and the C₁₂-C₂₄ acyl lysophosphatidylcholine,prevents and/or minimizes the aggregation that occurs when that compoundis omitted from the membrane.

Compositions

The invention provides lipid-based nanoparticles, and compositionscomprising the same. In certain embodiments, the nanoparticle comprises,and/or is defined by, a bipolar lipid membrane.

In certain embodiments, the membrane comprises cholesterol. In otherembodiments, the membrane comprises dicetyl phosphate. In yet otherembodiments, the membrane comprises an amphipathic lipid. In yet otherembodiments, the membrane comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In yet otherembodiments, the membrane comprises cholesterol, dicetyl phosphate, andDSPC. In yet other embodiments, the membrane comprises a hepatocytereceptor binding molecule.

In certain embodiments, the amphipathic lipid comprises at least oneselected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In other embodiments, theamphipathic lipid comprises at least one selected from the groupconsisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

In certain embodiments, the hepatocyte receptor binding moleculecomprises biotin. In other embodiments, the biotin-containing hepatocytereceptor binding molecule comprises at least one selected from the groupconsisting of N-hydroxysuccinimide (NHS) biotin; sulfo-NHS-biotin;N-hydroxysuccinimide long chain biotin; sulfo-N-hydroxysuccinimide longchain biotin; D-biotin; biocytin; sulfo-N-hydroxysuccinimide-S—S-biotin;biotin-BMCC; biotin-HPDP; iodoacetyl-LC-biotin; biotin-hydrazide;biotin-LC-hydrazide; biocytin hydrazide; biotin cadaverine;carboxybiotin; photobiotin; ρ-aminobenzoyl biocytin trifluoroacetate;ρ-diazobenzoyl biocytin; biotin DHPE (2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate); biotin-X-DHPE (2,3-diacetoxypropyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide;

biotinethylenediamine; biotin-XL; biotin-X-ethylenediamine; biotin-XXhydrazide; biotin-XX-SE; biotin-XX, SSE; biotin-X-cadaverine;α-(t-BOC)biocytin; N-(biotinyl)-N′-(iodoacetyl) ethylenediamine;DNP-X-biocytin-X-SE; biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+)biotin 4-amidobenzoic acid sodium salt;

Biotin 2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; and biotin6-O-phospho-α-D-mannopyranoside.

In certain embodiments, the hepatocyte receptor binding molecule isselected form the group consisting of 2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate (biotin DHPE) and biotin-X-DHPE (2,3-diacetoxy propyl2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate).

In certain embodiments, the cholesterol ranges from about 5% to about15% (w/w) in the membrane. In other embodiments, the cholesterol ispresent in the membrane at a concentration of about 5%, 5.5%, 6%, 6.5%,7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%,13.5%, 14%, 14.5%, or 15% (w/w).

In certain embodiments, the dicetyl phosphate ranges from about 10% toabout 25% (w/w) in the membrane. In other embodiments, the dicetylphosphate is present in the membrane ata concentration of about 10%,10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%,16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%,22.5%, 23%, 23.5%, 24%, 24.5%, or 25% (w/w).

In certain embodiments, the DSPC ranges from about 40% to about 75%(w/w) in the membrane. In other embodiments, the DSPC is present in themembrane at a concentration of about 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or75% (w/w).

In certain embodiments, the hepatocyte receptor binding molecule rangesfrom about 0.5% to about 4% (w/w) in the membrane. In other embodiments,the hepatocyte receptor binding molecule is present in the membrane at aconcentration of about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%,1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%,2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%,3.7%, 3.8%, 3.9%, or 4.0% (w/w).

In certain embodiments, the membrane comprises at least one compoundselected from the group consisting of a stabilizer and a C₁₂-C₂₄ acyllysophosphatidylcholine.

In certain embodiments, the membrane further comprises a C₁₂-C₂₄ acyllysophosphatidylcholine. In other embodiments, the membrane furthercomprises stearoyl lysophosphatidylcholine.

In certain embodiments, the membrane further comprises m-cresol.

In certain embodiments, the stabilizer is selected from the groupconsisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate,thiomersal, and butylated hydroxytoluene(2,6-di-tert-butyl-4-methylphenol).

In certain embodiments, the stabilizer ranges from about 10% to about25% (w/w) in the membrane. In other embodiments, the stabilizer ispresent in the membrane at a concentration of about 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w/w).

In certain embodiments, the m-cresol ranges from about 10% to about 25%(w/w) in the membrane. In other embodiments, the m-cresol is present inthe membrane at a concentration of about 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w/w).

In certain embodiments, the C₁₂-C₂₄ lysophosphatidylcholine ranges fromabout 5% to about 30% (w/w) in the membrane. In other embodiments, theC₁₂-C₂₄ lysophosphatidylcholine ranges from about 1% to about 30% (w/w)in the membrane. In yet other embodiments, the C₁₂-C₂₄lysophosphatidylcholine is present in the membrane at a concentration ofabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or30% (w/w).

In certain embodiments, the stearoyl lysophosphatidylcholine ranges fromabout 5% to about 30% (w/w) in the membrane. In other embodiments, thestearoyl lysophosphatidylcholine ranges from about 1% to about 30% (w/w)in the membrane. In yet other embodiments, the stearoyllysophosphatidylcholine is present in the membrane at a concentration ofabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or30% (w/w).

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1% to about 30% (w/w)of the amount of DSPC in the membrane. In yet other embodiments, theamount of the C₁₂-C₂₄ lysophosphatidylcholine in the membrane is about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29% (w/w) or 30% (w/w) of the amount of DSPC in the membrane.

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1 mole % to about 50mole % of the amount of DSPC in the membrane. In yet other embodiments,the amount of the C₁₂-C₂₄ lysophosphatidylcholine in the membrane isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50mole % of the amount of DSPC in the membrane.

In certain embodiments, the amount of the stearoyllysophosphatidylcholine in the membrane is about 1% to about 30% (w/w)of the amount of DSPC in the membrane. In yet other embodiments, theamount of the stearoyl lysophosphatidylcholine in the membrane is about1%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w/w) of theamount of DSPC in the membrane.

In certain embodiments, the amount of the stearoyllysophosphatidylcholine in the membrane is about 1 mole % to about 50mole % of the amount of DSPC in the membrane. In yet other embodiments,the amount of the stearoyl lysophosphatidylcholine in the membrane isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50mole % of the amount of DSPC in the membrane.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE. In other embodiments, the membrane comprises cholesterol,dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, andbiotin DHPE.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, m-cresol, and at least one selected from the groupconsisting of biotin DHPE and biotin-X-DHPE. In other embodiments, themembrane comprises cholesterol, dicetyl phosphate, DSPC, m-cresol, andbiotin DHPE.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and at least oneselected from the group consisting of biotin DHPE and biotin-X-DHPE. Inother embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and biotin DHPE.

In certain embodiments, the stabilizer is contacted with the membrane,and/or the lipid components that assemble to form the membrane (such as,but not limited to, cholesterol, dicetyl phosphate, DSPC, C₁₂-C₂₄lysophosphatidylcholine if present, and biotin DHPE), at a (w/w) ratioof the membrane to the stabilizer ranging from about 1:1 to about 1:30.In other embodiments, the stabilizer is contacted with the membrane,and/or the lipid components that assemble to form the membrane, at a(w/w) ratio of the membrane to the stabilizer of about 1:1, 1:1.5, 1:2,1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8,1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 or1:30.

In certain embodiments, the m-cresol is contacted with the membrane,and/or the lipid components that assemble to form the membrane (such as,but not limited to, cholesterol, dicetyl phosphate, DSPC, C₁₂-C₂₄lysophosphatidylcholine if present, and biotin DHPE), at a (w/w) ratioof the membrane to the stabilizer ranging from about 1:1 to about 1:30.In other embodiments, the m-cresol is contacted with the membrane,and/or the lipid components that assemble to form the membrane, at a(w/w) ratio of the membrane to the stabilizer of about 1:1, 1:1.5, 1:2,1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8,1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18,1:19, 1:20, 1:21, 1;22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29 or1:30.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotinDHPE, in a % (w/w) ratio of about 9.4:18.1:56.8:14.1:0.0:1.5.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and biotin DHPE, in a% (w/w) ratio of about 9.4:18.1:56.8:14.1:1.5.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotinDHPE, in a % (w/w) ratio of about 7.7:15.0:58.6:0.0:17.4:1.3.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, and biotin DHPE, in a % (w/w) ratio of about9.3:18.2:71.0:1.5.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and biotinDHPE, in a % (w/w) ratio of about 8.4:16.2:47.5:7.6:19.0:1.3.

In certain embodiments, the membrane comprises cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, and biotin DHPE, in a% (w/w) ratio of about 10.4:20:58.6:9.4:1.6.

In certain embodiments, the at least one hepatocyte receptor bindingmolecule extends outward from the nanoparticle.

The invention should not be construed to be limited to the constructsdescribed and/or exemplified herein. Rather, the invention providesmethods of stabilizing and/or preventing aggregation of liposomes andother lipid-based nanoparticles, wherein the membrane is contacted withat least one selected from the group consisting of a stabilizer and aC₁₂-C₂₄ acyl lysophosphatidylcholine. In certain embodiments, thecontacting removes or minimizes any “free” edges in the membrane thatlead to aggregation of the liposomes and other lipid-basednanoparticles.

In certain embodiments, the stabilizer is selected from the groupconsisting of m-cresol, benzyl alcohol, methyl 4-hydroxybenzoate,thiomersal, and butylated hydroxytoluene. In other embodiments, thestabilizer, such as but not limited to m-cresol, ranges from about 10%to about 25% (w/w) in the membrane. In yet other embodiments, thestabilizer, such as but not limited to m-cresol, is present in themembrane at a concentration of about 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% (w/w).

In certain embodiments, the C₁₂-C₂₄ lysophosphatidylcholine, such as butnot limited to stearoyl lysophosphatidylcholine, ranges from about 5% toabout 30% (w/w) in the membrane. In other embodiments, the C₁₂-C₂₄lysophosphatidylcholine, such as but not limited to stearoyllysophosphatidylcholine, ranges from about 1% to about 30% (w/w) in themembrane. In yet other embodiments, the C₁₂-C₂₄ lysophosphatidylcholine,such as but not limited to stearoyl lysophosphatidylcholine, is presentin the membrane at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% (w/w).

In certain embodiments, the membrane comprises at least one amphipathiclipid selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In other embodiments, theamphipathic lipid is at least one selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1%-30% (w/w) of theamount of the at least one amphipathic lipid in the membrane. In yetother embodiments, the amount of the C₁₂-C₂₄ lysophosphatidylcholine inthe membrane is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29% or 30% (w/w) of the amount of the at least one amphipathiclipid in the membrane.

In certain embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1 mole % to about 50mole % of the amount of the at least one amphipathic lipid in themembrane. In yet other embodiments, the amount of the C₁₂-C₂₄lysophosphatidylcholine in the membrane is about 1, 2, 3, 4, 5, 6, 7, 8,9, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mole % of the amount of the atleast one amphipathic lipid in the membrane.

In certain embodiments, the stabilizer, such as but not limited tom-cresol, is contacted with the membrane, and/or the lipid componentsthat assemble to form the membrane, at a (w/w) ratio ranging from about1:1 to about 1:30. In other embodiments, the stabilizer, such as but notlimited to m-cresol, is contacted with the membrane, and/or the lipidcomponents that assemble to form the membrane, ata (w/w) ratio of about1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5,1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15,1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1;22, 1:23, 1:24, 1:25, 1:26, 1:27,1:28, 1:29 or 1:30.

In certain embodiments, the size of the nanoparticle ranges from about10 nm to about 150 nm. In other embodiments, the size of thenanoparticle is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm,80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm or 150 nm.

In certain embodiments, a therapeutic agent is dispersed within and/oradsorbed onto the nanoparticle. In other embodiments, the therapeuticagent is covalently bound to the nanoparticle. In yet other embodiments,the therapeutic agent is not covalently bound to the nanoparticle.

In certain embodiments, the therapeutic agent comprises at least oneselected from the group consisting of insulin, insulin analogs, amylin,interferon, parathyroid hormone, calcitonin, serotonin, serotoninagonist, serotonin reuptake inhibitor, human growth hormone, GIP,anti-GIP monoclonal antibody, metformin, bromocriptine, dopamine,glucagon and GLP-1. In other embodiments, the therapeutic agent isinsulin.

In certain embodiments, the nanoparticle is suspended in an aqueoussolution comprising a free dissolved therapeutic agent that is notdispersed within the nanoparticle.

In certain embodiments, the nanoparticle-dispersed insulin and the freedissolved insulin are independently selected from the group consistingof insulin lispro, insulin aspart, regular insulin, insulin glargine,insulin zinc, extended human insulin zinc suspension, isophane insulin,human buffered regular insulin, insulin glulisine, recombinant humanregular insulin, and recombinant human insulin isophane.

In certain embodiments, the lipid further comprises cellulose acetatephthalate. In other embodiments, the cellulose acetate phthalate is atleast partially bound to the therapeutic agent dispersed within thenanoparticle.

In certain embodiments, at least one charged organic molecule is boundto the therapeutic agent dispersed within the nanoparticle. In otherembodiments, the charged organic molecule is at least one selected fromthe group consisting of protamines, polylysine, poly (arg-pro-thr)n in amole ratio of 1:1:1, poly (DL-Ala-poly-L-lys)n in a mole ratio of 6:1,histones, sugar polymers comprising a primary amino group,polynucleotides with primary amino groups, proteins comprising aminoacid residues with carboxyl (COO⁻) or sulfhydral (S⁻) functional groups,and acidic polymers (such as sugar polymers containing carboxyl groups).

In certain embodiments, the nanoparticle of the invention, andcompositions comprising the same, help deliver the therapeutic agentdispersed therewithin to the hepatocytes in the liver.

In certain embodiments, the compositions of the invention comprise aneffective dose of a hepatocyte targeted pharmaceutical composition thatcombines free therapeutic drug (such as, but not limited to, insulin)and therapeutic drug associated with the lipid-based nanoparticle of theinvention. The combination of free therapeutic drug and therapeutic drugassociated with the lipid-based nanoparticle creates a dynamicequilibrium process between the two forms of therapeutic drug thatoccurs in vivo to help control the movement of free therapeutic drug tothe receptor sites of hormonal action. In the case of insulin as thetherapeutic drug, those receptor sites are the muscle and adiposetissues of a diabetic patient. Hepatocyte targeted therapeutic drug isalso delivered to the liver of a patient over a different designatedtime period than free therapeutic drug, thereby introducing newpharmacodynamic profiles of therapeutic drug when the therapeutic drugremains associated with the nanoparticle and/or when free therapeuticdrug is released from the nanoparticle. In addition, a portion oftherapeutic drug that is associated with the nanoparticle is targeted tothe liver. In the case of insulin as the therapeutic drug, the newpharmacodynamic profile of the product provides not only basal insulinfor peripheral tissues, but also meal-time hepatic therapeutic drugstimulation for the management of hepatic glucose storage during a meal.Free insulin is released from the site of administration and isdistributed throughout the body. Insulin associated with the lipid-basednanoparticle is delivered to the liver. The rate of release of insulinassociated with the nanoparticle is different than the rate of releaseof free insulin from the site of administration. These different releaserates of insulin delivery, combined with the targeted delivery ofinsulin associated with the nanoparticle to the liver, provide for thenormalization of glucose concentrations in patients with Type I and TypeII diabetes mellitus. In certain embodiments, the hepatocyte targetedcomposition comprises any therapeutically effective insulin or insulinderivative or analog, or any combination of two or more types of insulinor insulin derivative or analog.

Compounds described herein also include isotopically labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In certain embodiments, isotopically labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In other embodiments, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet otherembodiments, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In certain embodiments, the compounds described herein are labeled byother means, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

Compounds of the invention can in certain embodiments form acids orbases. In certain embodiments, the invention contemplates acid additionsalts. In other embodiments, the invention contemplates base additionsalts. In yet other embodiments, the invention contemplatespharmaceutically acceptable acid addition salts. In yet otherembodiments, the invention contemplates pharmaceutically acceptable baseaddition salts. Pharmaceutically acceptable salts refer to salts ofthose bases or acids that are not toxic or otherwise biologicallyundesirable.

Suitable pharmaceutically acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids(including hydrogen phosphate and dihydrogen phosphate). Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,araliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which include formic, acetic, propionic, succinic,glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic,glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include, for example, metallic salts including alkalimetal, alkaline earth metal and transition metal salts such as, forexample, calcium, magnesium, potassium, sodium, lithium and copper, ironand zinc salts. Pharmaceutically acceptable base addition salts alsoinclude organic salts made from basic amines such as, for example,N,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. All ofthese salts may be prepared from the corresponding compound by reacting,for example, the appropriate acid or base with the compound.

Disclosed is a kit comprising any composition of the invention and aninstructional material which describes administering the composition toa tissue of a subject, such as a mammal. This kit may comprise a(preferably sterile) solvent suitable for dissolving or suspending thecomposition of the invention prior to administering the composition tothe subject, such as a mammal.

Methods

The invention provides methods of preparing the lipid-based nanoparticleof the invention. In certain embodiments, the method comprisescontacting in an aqueous system cholesterol, dicetyl phosphate,amphipathic lipid, hepatocyte receptor binding molecule, and at leastone compound selected from the group consisting of a stabilizer andstearoyl lysophosphatidylcholine. In other embodiments, the methodcomprises contacting in an aqueous system cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol andbiotin-DHPE.

In certain embodiments, the stabilizer is added to the aqueous systemafter the cholesterol, dicetyl phosphate, amphipathic lipid, optionallystearoyl lysophosphatidylcholine, and hepatocyte receptor bindingmolecule had been contacted in the aqueous system.

In certain embodiments, the m-cresol is added to the aqueous systemafter the cholesterol, dicetyl phosphate, DSPC, stearoyllysophosphatidylcholine and hepatocyte receptor binding molecule hadbeen contacted in the aqueous system.

In certain embodiments, the nanoparticle comprises a therapeutic agentdispersed therewithin.

In certain embodiments, the therapeutic agent, cholesterol, dicetylphosphate, amphipathic lipid, hepatocyte receptor binding molecule, andthe at least one compound are simultaneously contacted in the aqueoussystem.

In certain embodiments, the therapeutic agent, cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol andbiotin-DHPE are simultaneously contacted in the aqueous system.

In certain embodiments, the nanoparticle is formed in the absence of thetherapeutic agent, wherein optionally the nanoparticle is at leastpartially concentrated, purified or isolated, and wherein thetherapeutic agent is contacted with the nanoparticle, whereby at least aportion of the therapeutic agent is dispersed within the nanoparticle.

In certain embodiments, the composition is treated with celluloseacetate phthalate, which can bind non-covalently to at least a portionof the therapeutic agent dispersed within the nanoparticle and protectthe therapeutic agent from metabolic degradation. In other embodiments,the cellulose acetate phthalate is covalently bound to the therapeuticagent and/or any of the lipids that constitute the nanoparticle.

Further embodiments relating to certain methods for preparing and/orprocessing and/or purifying a nanoparticle can be found for examplelimited in U.S. Patent Application Nos. US20110135725 and US20090087479,all of which are incorporated herein in their entireties by reference.

The invention further provides a method of treating a disease in amammal. In certain embodiments, the method comprises administering tothe mammal in need thereof a therapeutically effective amount of ananoparticle and/or a composition of the invention.

In certain embodiments, the disease is diabetes mellitus and thetherapeutic agent comprises insulin.

The invention further provides a method of activating hepatic glycogensynthase in a mammal. In certain embodiments, the method comprisesadministering to the mammal in need thereof a therapeutically effectiveamount of a nanoparticle and/or composition of the invention, whereinthe therapeutic agent comprises insulin. In other embodiments, themammal has diabetes mellitus.

Administration/Dosage/Formulations

The invention also encompasses pharmaceutical compositions and methodsof their use. These pharmaceutical compositions may comprise an activeingredient (which can be one or more compositions of the invention, orpharmaceutically acceptable salts thereof) optionally in combinationwith one or more pharmaceutically acceptable agents. The compositionsset forth herein can be used alone or in combination with additionalcompounds to produce additive, complementary, or synergistic effects.

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the onset of a disease or disorder contemplatedherein. Further, several divided dosages, as well as staggered dosagesmay be administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection, or may beadministered inhalationally, buccally and/or orally. Further, thedosages of the therapeutic formulations may be proportionally increasedor decreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder contemplated herein. An effective amountof the therapeutic compound necessary to achieve a therapeutic effectmay vary according to factors such as the state of the disease ordisorder in the patient; the age, sex, and weight of the patient; andthe ability of the therapeutic compound to treat a disease or disordercontemplated herein. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A non-limitingexample of an effective dose range for a therapeutic compound of theinvention is from about 1 and 5,000 mg/kg of body weight/per day. One ofordinary skill in the art would be able to study the relevant factorsand make the determination regarding the effective amount of thetherapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect, andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease or disorder contemplated herein.

In certain embodiments, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Incertain embodiments, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils, as long as the solvent or dispersion mediumdoes not disrupt the nanoparticle significantly. Prevention of theaction of microorganisms may be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it is preferableto include isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions may be brought aboutby including in the composition an agent that delays absorption, forexample, aluminum monostearate or gelatin.

In certain embodiments, the compositions of the invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of the inventionare administered to the patient in range of dosages that include, butare not limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from individual toindividual depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient is determined by the attending physicaltaking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 350 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial increments therebetween.

In certain embodiments, the dose of a compound and/or composition of theinvention is from about 1 mg and about 2,500 mg. In other embodiments, adose of a compound of the invention used in compositions describedherein is less than about 10,000 mg, or less than about 8,000 mg, orless than about 6,000 mg, or less than about 5,000 mg, or less thanabout 3,000 mg, or less than about 2,000 mg, or less than about 1,000mg, or less than about 500 mg, or less than about 200 mg, or less thanabout 50 mg. Similarly, in other embodiments, a dose of a secondcompound as described herein is less than about 1,000 mg, or less thanabout 800 mg, or less than about 600 mg, or less than about 500 mg, orless than about 400 mg, or less than about 300 mg, or less than about200 mg, or less than about 100 mg, or less than about 50 mg, or lessthan about 40 mg, or less than about 30 mg, or less than about 25 mg, orless than about 20 mg, or less than about 15 mg, or less than about 10mg, or less than about 5 mg, or less than about 2 mg, or less than about1 mg, or less than about 0.5 mg, and any and all whole or partialincrements thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound and/or composition of theinvention, alone or in combination with a second pharmaceutical agent;and instructions for using the compound to treat, prevent, or reduce oneor more symptoms of a disease or disorder contemplated herein.

In certain embodiments, the container holds a lipid-based nanoparticle,which does not comprise a therapeutic agent of interest, such as but notlimited to an insulin or a derivative or analog thereof. In otherembodiments, the container holds a lipid-based nanoparticle, whichcomprises a therapeutic agent of interest, such as but not limited to aninsulin or a derivative or analog thereof. In yet other embodiments, thecontainer further holds a therapeutic agent of interest, such as but notlimited to an insulin or a derivative or analog thereof.

Illustrative Non-Limiting Methods of Treating Diabetes Mellitus

Patients with Type I or Type II diabetes mellitus can be administered aneffective amount of a nanoparticle of the invention comprising aninsulin. When this composition is administered subcutaneously, a portionof the composition enters the circulatory system where the compositionis transported to the liver and other areas where the extendedamphipathic lipid binds the lipid construct to receptors of hepatocytes.A portion of the administered composition is exposed to an externalgradient in vivo, where insulin can be solubilized and then move fromthe lipid construct thereby supplying insulin to the muscle and adiposetissue. Insulin that remains with the lipid construct maintains thecapability of being directed to the hepatocyte binding receptor on thehepatocytes in the liver. Therefore, two forms of insulin are producedfrom this particular lipid construct. In an in vivo setting, free andlipid associated insulin are generated in a time-dependent manner.

Administration of the nanoparticles and compositions comprising same canbe through any of the accepted modes of administration for insulin thatare desired to be administered. These methods include oral, parenteral,nasal and other systemic or aerosol forms. These methods further includepump delivery systems.

Oral administration of a nanoparticle of the invention is followed byintestinal absorption of insulin associated with the nanoparticle of theinvention into the circulatory system of the body, where it is alsoexposed to the physiological pH of the blood. The nanoparticle istargeted for delivery to the liver, and may be shielded by the presenceof cellulose acetate phthalate within the nanoparticle of the invention.In the case of oral administration, the shielded nanoparticletransverses the oral cavity, migrates through the stomach and moves intothe small intestine, where the alkaline pH of the small intestinedegrades the cellulose acetate phthalate shield. The deshieldednanoparticle is absorbed into the circulatory system. This enables thenanoparticle to be delivered to the sinusoids of the liver. A receptorbinding molecule, such as1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) or anyother hepatocyte specific molecule, provides a means for lipid constructto bind to the receptor and then be engulfed or endocytosed by thehepatocytes. Insulin is then released from the nanoparticle where, upongaining access to the cellular environment, it performs its designatedfunction with regard to acting as an agent to control diabetes mellitus.

Patients with Type I or Type II diabetes mellitus may be administered aneffective amount of a nanoparticle comprising a mixture of free glargineinsulin and glargine insulin associated with the nanoparticle. Glargineinsulin can be combined with other forms of insulin, such as insulinlispro, insulin aspart, regular insulin, insulin zinc, human insulinzinc extended, isophane insulin, human buffered regular insulin, insulinglulisine, recombinant human regular insulin, recombinant human insulinisophane or premixed combinations of any of the aforementioned insulins,a derivative thereof, and a combination of any of the aforementionedinsulins. The composition can be administered by a subcutaneous or oralroute.

After a composition is administered to a patient by subcutaneousinjection, the in situ physiological environment in the injection area,the morphology and chemical structures of free insulin and the insulinassociated with the nanoparticle begin to change. For example, as the pHof the environment around the free glargine insulin and the glargineinsulin associated with the nanoparticle increases after being dilutedwith physiological media, the pH reaches the isoelectric point ofglargine insulin, where flocculation, aggregation and precipitationreactions occur for both free glargine insulin and glargine insulinassociated with the nanoparticle. In certain embodiments, free glargineinsulin changes from a soluble form at injection, to a insoluble form ata pH near its isoelectric point of pH 5.8-6.2, and then to a solubleform at physiological pH. The rates at which these processes occurdiffer between free glargine insulin and glargine insulin associatedwith the nanoparticle. The free glargine insulin is directly exposed tochanges in pH and dilution. Exposure of glargine insulin associated withthe nanoparticle to small changes in pH and dilution at physiological pHis delayed due to the time required for diffusion of physiologicalfluids or media through the lipid bilayer in the nanoparticle. The delayin the release of insulin from the lipid construct as well as the delayof the release of the insulin associated with the nanoparticle is afeature of the invention since it affects and augments the biologicaland pharmacological response in vivo.

Oral administration of a pharmaceutical composition that combines freeglargine insulin and glargine insulin associated with a nanoparticle isfollowed by intestinal absorption of glargine insulin associated withthe nanoparticle into the circulatory system of the body, where it isalso exposed to the physiological pH of the blood. In certainembodiments, the composition comprises a delayed release matrix whichreleases HDV glargine over a prolonged period of time, in order toachieve a 24-hour dose regimen. All or a portion of the nanoparticle isdelivered to the liver.

Patients with Type I or Type II diabetes mellitus can be administered aneffective amount of a hepatocyte targeted composition comprising amixture of free recombinant human insulin isophane (NPH) plus freerecombinant human regular insulin along with recombinant human insulinisophane and recombinant human regular insulin which are both associatedwith a nanoparticle. Recombinant human insulin isophane can be combinedwith other forms of insulin, such as insulin lispro, insulin aspart,regular insulin, insulin glargine, insulin zinc, human insulin zincextended, isophane insulin, human buffered regular insulin, insulinglulisine, recombinant human regular insulin, recombinant human insulinisophane, or any (premixed) combinations thereof.

In certain embodiments, the composition comprises a delayed releasematrix which releases HDV NPH over a prolonged period of time, in orderto achieve a 24-hour dose regimen.

Oral administration of a pharmaceutical composition that combines freerecombinant human insulin isophane and recombinant human insulinisophane associated with a nanoparticle is followed by intestinalabsorption of recombinant human insulin isophane associated with thenanoparticle into the circulatory system of the body where it is alsoexposed to the physiological pH of the blood. All or a portion of thenanoparticle is delivered to the liver, while the non-HDV isophane isslowly absorbed from a slow release matrix for release into the generalcirculation.

As the physiological dilution is increased in situ in the subcutaneousspace or upon entering into the circulatory system, free recombinanthuman insulin isophane and recombinant human insulin isophane associatedwith the nanoparticle encounter a normal physiological pH environment ofpH 7.4. As a result of dilution free recombinant human insulin isophanechanges from an insoluble form at injection, to a soluble form atphysiological pH. In the soluble form, recombinant human insulinisophane migrates through the body to sites where it is capable ofeliciting a pharmacological response. Recombinant human insulin isophaneassociated with the nanoparticle becomes solubilized and released fromthe nanoparticle at a different rate that is slower than that of freerecombinant human insulin isophane. This is because recombinant humaninsulin isophane associated with the nanoparticle has to traverse thecore volume and lipid domains of the nanoparticle before it contacts thebulk phase media.

The amount of insulin administered will be dependent on the subjectbeing treated, the type and severity of the affliction, the manner ofadministration and the judgment of the prescribing physician. Althougheffective dosage ranges for specific biologically active substances ofinterest are dependent upon a variety of factors, and are generallyknown to one of ordinary skill in the art, some dosage guidelines can begenerally defined. For most forms of administration, the nanoparticlewill be suspended in an aqueous solution and generally not exceed 4.0%(w/v) of the total formulation. The drug component of the formulationwill in certain embodiments be less than 20% (w/v) of the formulationand generally greater than 0.01% (w/v).

In certain embodiments, the pharmaceutical composition comprises HDVinsulin, and no free insulin. In such cases, all of the insulin withinthe composition is targeted to the liver. In other embodiments, thepharmaceutical composition comprises HDV insulin and free insulin(non-HDV insulin). The ratio between HDV insulin and free insulin canbe, in non-limiting example, about 0.1:99.9, 0.2:99.8, 0.3:99.7,0.4:99.6, 0.5:99.5, 0.6:99.4, 0.7:99.3, 0.8:99.2, 0.9:99.1, 1:99, 2:98,3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91, 10:90, 12:88, 14:86, 16:84,18:82, 20:80, 22:78, 24:76, 25:75, 26:74, 28:72, 30:70, 32:68, 34:66,36:64, 38:62, 40:60, 42:58, 44:56, 46:54, 48:52, and/or 50:50.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 5% with the balance made up from non-toxic carriers can beprepared.

The exact composition of these formulations may vary widely depending onthe particular properties of the drug in question. In certainembodiments, they comprise from 0.01% to 5%, and preferably from 0.05%to 1% active ingredient for highly potent drugs, and from 2%-4% formoderately active drugs.

The percentage of active ingredient contained in such parenteralcompositions is highly dependent on the specific nature thereof, as wellas the activity of the active ingredient and the needs of the subject.However, percentages of active ingredient of 0.01% to 5% in solution areemployable, and will be higher if the composition is a solid which willbe subsequently diluted to the above percentages. In certainembodiments, the composition comprises 0.2%-2.0% of the active agent insolution.

Administration

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents. Routes of administration of any of thecompositions of the invention include oral, nasal, rectal, intravaginal,parenteral, buccal, sublingual or topical. The compounds and/orcompositions for use in the invention may be formulated foradministration by any suitable route, such as for oral or parenteral,for example, transdermal, transmucosal (e.g., sublingual, lingual,(trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compounds and/or compositions of theinvention may be in the form of tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropyl methylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds and/orcompositions of the invention, and a further layer providing for theimmediate release of a medication for treatment of diseases ordisorders. Using a wax/pH-sensitive polymer mix, a gastric insolublecomposition may be obtained in which the active ingredient is entrapped,ensuring its delayed release.

Parenteral Administration

For parenteral administration, the compounds and/or compositions of theinvention may be formulated for injection or infusion, for example,intravenous, intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Pulmonary Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 microns, and preferably from about 1 to about6 microns. Such compositions are conveniently in the form of dry powdersfor administration using a device comprising a dry powder reservoir towhich a stream of propellant may be directed to disperse the powder orusing a self-propelling solvent/powder-dispensing container such as adevice comprising the active ingredient dissolved or suspended in alow-boiling propellant in a sealed container. Preferably, such powderscomprise particles wherein at least 98% of the particles by weight havea diameter greater than 0.5 microns and at least 95% of the particles bynumber have a diameter less than 7 microns. More preferably, at least95% of the particles by weight have a diameter greater than 1 nanometerand at least 90% of the particles by number have a diameter less than 6microns. Dry powder compositions preferably include a solid fine powderdiluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile for administration by injection,comprising the active ingredient, and may conveniently be administeredusing any nebulization or atomization device. In certain embodiments,the compounds and/or compositions of the invention are sterile filteredbefore administration to the subject. Such formulations may furthercomprise one or more additional ingredients including, but not limitedto, a flavoring agent such as saccharin sodium, a volatile oil, abuffering agent, a surface active agent, or a preservative such asmethylhydroxybenzoate. The droplets provided by this route ofadministration preferably have an average diameter in the range fromabout 0.1 to about 200 microns.

Intranasal Delivery

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 microns. Such a formulation is administered in themanner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 75% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

Buccal Delivery

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active ingredient, the balance comprising an orally dissolvable ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder or an aerosolized oratomized solution or suspension comprising the active ingredient. Suchpowdered, aerosolized, or aerosolized formulations, when dispersed,preferably have an average particle or droplet size in the range fromabout 0.1 to about 200 microns, and may further comprise one or more ofthe additional ingredients described herein.

Ophthalmic Administration

A pharmaceutical composition of the invention may be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1%-1.0% (w/w) solution or suspension of the activeingredient in an aqueous or oily liquid carrier. Such drops may furthercomprise buffering agents, salts, or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form or in a lipid construct preparation.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release that is longer that the same amount of agent administeredin bolus form.

For sustained release, the compositions may be formulated with asuitable polymer or hydrophobic material that provides sustained releaseproperties to the compounds and/or compositions. As such, thecompositions and/or compositions for use the method of the invention maybe administered in the form of microparticles, for example, by injectionor in the form of wafers or discs by implantation.

In certain embodiments, the compounds and/or compositions of theinvention are administered to a patient, alone or in combination withanother pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound and/orcomposition of the present invention depends on the age, sex and weightof the patient, the current medical condition of the patient and theprogression of a disease or disorder contemplated herein in the patientbeing treated. The skilled artisan is able to determine appropriatedosages depending on these and other factors.

A suitable dose of a compound and/or composition of the presentinvention may be in the range of from about 0.01 mg to about 5,000 mgper day, such as from about 0.1 mg to about 1,000 mg, for example, fromabout 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day.The dose may be administered in a single dosage or in multiple dosages,for example from 1 to 4 or more times per day. When multiple dosages areused, the amount of each dosage may be the same or different. Forexample, a dose of 1 mg per day may be administered as two 0.5 mg doses,with about a 12-hour interval between doses.

It is understood that the amount of compound and/or composition dosedper day may be administered, in non-limiting examples, every day, everyother day, every 2 days, every 3 days, every 4 days, or every 5 days.For example, with every other day administration, a 5 mg per day dosemay be initiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of theviral load, to a level at which the improved disease is retained. Incertain embodiments, patients require intermittent treatment on along-term basis upon any recurrence of symptoms and/or infection.

The compounds and/or compositions for use in the method of the inventionmay be formulated in unit dosage form. The term “unit dosage form”refers to physically discrete units suitable as unitary dosage forpatients undergoing treatment, with each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, optionally in association with a suitablepharmaceutical carrier. The unit dosage form may be for a single dailydose or one of multiple daily doses (e.g., about 1 to 4 or more timesper day). When multiple daily doses are used, the unit dosage form maybe the same or different for each dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compounds and/orcompositions lies preferably within a range of circulatingconcentrations that include the ED₅₀ with minimal toxicity. The dosageoptionally varies within this range depending upon the dosage formemployed and the route of administration utilized.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which the invention belongs. Generally, thenomenclature used herein and the laboratory procedures in organicchemistry and protein chemistry are those well known and commonlyemployed in the art.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” is understood by persons of ordinaryskill in the art and varies to some extent on the context in which it isused. As used herein when referring to a measurable value such as anamount, a temporal duration, and the like, the term “about” is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein, the term “active ingredient” refers to a therapeuticagent that is to be delivered to a subject to produce a therapeuticeffect in the subject. Non-limiting examples of active ingredientscontemplated within the invention are insulin, interferon, parathyroidhormone, calcitonin, serotonin, serotonin agonist, serotonin reuptakeinhibitor, human growth hormone, GIP, anti-GIP monoclonal antibody,metformin, bromocriptine, dopamine, glucagon and/or GLP-1.

The term “amphipathic lipid” means a lipid molecule having a polar andnon-polar end.

By “aqueous media” is meant water or water containing buffer or salt.

The term “bioavailability” refers to a measurement of the rate andextent that insulin reaches the systemic circulation and is available atthe sites of action.

In one aspect, the terms “co-administered” and “co-administration” asrelating to a subject refer to administering to the subject a compoundof the invention or salt thereof along with a compound that may alsotreat any disease or disorder contemplated herein and/or with a compoundthat is useful in treating other medical conditions but which inthemselves may cause or facilitate any disease or disorder contemplatedherein. In certain embodiments, the co-administered compounds areadministered separately, or in any kind of combination as part of asingle therapeutic approach. The co-administered compound may beformulated in any kind of combinations as mixtures of solids and liquidsunder a variety of solid, gel, and liquid formulations, and as asolution.

As used herein, a “disease” is a state of health of a subject whereinthe subject cannot maintain homeostasis, and wherein if the disease isnot ameliorated then the subject's health continues to deteriorate.

As used herein, a “disorder” in a subject is a state of health in whichthe subject is able to maintain homeostasis, but in which the subject'sstate of health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the subject's state of health.

As used herein, the term “ED₅₀” refers to the effective dose of aformulation that produces 50% of the maximal effect in subjects that areadministered that formulation.

As used herein, an “effective amount,” “therapeutically effectiveamount” or “pharmaceutically effective amount” of a compound is thatamount of compound that is sufficient to provide a beneficial effect tothe subject to which the compound is administered.

The term “free active ingredient” or “free therapeutic agent” refers toan active ingredient or therapeutic agent that is not dispersed withinthe lipid particle (i.e., located within, adsorbed on and/or bound tothe lipid particle membrane).

The terms “glargine” and “glargine insulin” both refer to a recombinanthuman insulin analog which differs from human insulin in that the aminoacid asparagine at position A21 is replaced by glycine and two argininesare added to the C-terminus of the B-chain. Chemically, it is21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has theempirical formula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expressionthat can be used to communicate the usefulness of the composition and/orcompound of the invention in a kit. The instructional material of thekit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container that contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

The term “insulin” refers to natural or recombinant forms of insulin,and derivatives of the aforementioned insulins. Examples of insulininclude, but are not limited to insulin lispro, insulin aspart (such as,for example, FIASP®, Novo Nordisk), regular insulin, insulin glargine,insulin zinc, human insulin zinc extended, isophane insulin, humanbuffered regular insulin, insulin glulisine, recombinant human regularinsulin, and recombinant human insulin isophane. Also included areanimal insulins, such as bovine or porcine insulin.

The term “isoelectric point” refers to the pH at which theconcentrations of positive and negative charges on the protein are equaland, as a result, the protein will express a net zero charge. At theisoelectric point, a protein will exist almost entirely in the form of azwitterion, or hybrid between forms of the protein. Proteins are leaststable at their isoelectric points, and are more easily coagulated orprecipitated at this pH. However, proteins are not denatured uponisoelectric precipitation since this process is essentially reversible.

The term “lipid construct” refers to a lipid and/or phospholipidparticle in which individual lipid molecules interact to create abipolar lipid membrane that defines the boundaries of the lipidconstruct.

As the term is used herein, “to modulate” or “modulation of” abiological or chemical process or state refers to the alteration of thenormal course of the biological or chemical process, or changing thestate of the biological or chemical process to a new state that isdifferent than the present state. For example, modulation of theisoelectric point of a polypeptide may involve a change that increasesthe isoelectric point of the polypeptide. Alternatively, modulation ofthe isoelectric point of a polypeptide may involve a change thatdecreases the isoelectric point of a polypeptide.

The term “non-glargine insulin” refers at all insulins, either naturalor recombinant that are not glargine insulin. The term includesinsulin-like moieties, including fragments of insulin molecules, thathave biological activity of insulins.

As used herein, the term “pharmaceutical composition” or “composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a subject.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound useful within theinvention, and is relatively non-toxic, i.e., the material may beadministered to a subject without causing undesirable biological effectsor interacting in a deleterious manner with any of the components of thecomposition in which it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the subject such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the subject. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the subject.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compound prepared from pharmaceuticallyacceptable non-toxic acids and bases, including inorganic acids,inorganic bases, organic acids, inorganic bases, solvates, hydrates, andclathrates thereof.

The term “prevent,” “preventing” or “prevention,” as used herein, meansavoiding or delaying the onset of symptoms associated with a disease orcondition in a subject that has not developed such symptoms at the timethe administering of an agent or compound commences. Disease, conditionand disorder are used interchangeably herein.

By the term “specifically bind” or “specifically binds,” as used herein,is meant that a first molecule preferentially binds to a second molecule(e.g., a particular receptor or enzyme), but does not necessarily bindonly to that second molecule.

As used herein, a “subject” may be a human or non-human mammal or abird. Non-human mammals include, for example, livestock and pets, suchas ovine, bovine, porcine, canine, feline and murine mammals. In certainembodiments, the subject is human.

The term “treat,” “treating” or “treatment,” as used herein, meansreducing the frequency or severity with which symptoms of a disease orcondition are experienced by a subject by virtue of administering anagent or compound to the subject.

Throughout this disclosure, various aspects of the invention may bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range and, when appropriate,partial integers of the numerical values within ranges. For example,description of a range such as from 1 to 6 should be considered to havespecifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5,5.3, and 6. This applies regardless of the breadth of the range.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents are considered to be within the scope of this inventionand covered by the claims appended hereto. For example, it should beunderstood, that modifications in reaction conditions, including but notlimited to reaction times, reaction size/volume, and experimentalreagents, such as solvents, catalysts, pressures, atmosphericconditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents,with art-recognized alternatives and using no more than routineexperimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application. The following examples further illustrate aspectsof the present invention. However, they are in no way a limitation ofthe teachings or disclosure of the present invention as set forthherein.

EXPERIMENTAL EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, point out specific embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

The materials and methods used in the experiments presented in thisExperimental Example are now described.

Example 1

The compositions recited in Table 1 were prepared and characterized:

TABLE 1 Formulation (mg/mL) Component A B C D Distearoylphosphatidylcholine (DSPC) 10.075 8.063 10.075 7.53 Dicetyl phosphate(DCP) 2.575 2.575 2.575 2.57 Cholesterol 1.330 1.330 1.330 1.33 BiotinPE 0.220 0.220 0.220 0.21 Stearoyl lysophosphatidylcholine 0 2.0 0 1.21m-creosol 0 0 3.000 3.00

The amphipathic compounds were solubilized in a chloroform/methanol(2:1) mixture, followed by removal of organic solvents underrotoevaporization and vacuum. The dried material was then hydrated withthe phosphate buffer and homogenized to a particle size of less than 100nm.

Three formulations are illustrated in Table 1. Formulation A is acontrol nanoparticle formulation, which affords characteristic smallparticle size. The particle size determined for Formulation A was <100nm. Interestingly, within 2 weeks of its preparation, Formulation A wasfound to form wispy floater structures, which are disruptable aggregatesof individual nanoparticles (FIGS. 1A-1B). The nanoparticle aggregatesunderwent small increases in size over the period of several months.

In Formulation B, 25% of DSPC was replaced with stearoyllysophosphatidylcholine. The particle size determined for Formulation Bwas <100 nm. The particles formed in Formulation B were comparable insize to those in Formulation A, but Formulation B did not form wispystructures such as those observed with Formulation A.

Formulation C has the same amount of DSPC as Formulation A, but furthercomprises 3% (wt) m-creosol. The m-cresol is incorporated in a manner toprevent the aggregation of the nanoparticles, and Formulation C indeeddoes not form wispy structures such as those in Formulation A. Otherphenolic structures that have similar structures to m-cresol can be usedto stabilize the nanoparticles.

In certain embodiments, the stearoyl lysophosphatidylcholine and/or atleast one phenolic compound can be added to the initial amphipathicmixture before homogenization. In other embodiments, the at least onephenolic compound can be added after homogenization. Without wishing tobe limited by any theory, the at least one phenolic compound adsorbsinto the membrane edges to stabilize the particles.

Example 2: Bio-Efficacy of Nanoparticles with Stabilized Edges

Two versions of hepatocyte targeted nanoparticles comprising insulinlispro were tested in insulin deficient dogs to determine their efficacyas hypoglycemic agents. Formulation A was compared to a non-limitingexample of a formulation of the invention (Formulation B). As a controlstudy, a commercial lispro insulin lacking any hepatocyte targeting wasused. The dogs were maintained in a recognized animal research facilitywith all appropriate regulatory controls in place. Concentrations ofnanoparticles, doses of lispro insulin, food consumption and timing ofthe study were all comparable in this cross-over study.

In this open label cross-over study, beagle male dogs weighing 5-10 kgwere made insulin deficient with streptozotocin treatment. After beingstabilized on parenteral insulin injections and standardized feeding ofcontrol diets, the dogs were fasted overnight. The dog's blood glucoselevels were <200 mg/dL/kg body weight to be used for the study.

The dogs were fed a prescribed amount of standard dog diet 30 minutesfollowing a subcutaneous injection of lispro insulin in variousformulations: hepatocyte-targeted lispro insulin (Formulation A),hepatocyte-targeted lispro insulin (Formulation B), or commercial lisproinsulin lacking any hepatocyte targeting (control). Formulations A and Bhad similar effects in the oral glucose tolerance test (FIG. 2), showinga marked lowering of blood glucose levels following the test meal in thedogs as compared to an identical insulin dose with control lisproinsulin. Further, Formulation B showed an overall better performance inthis tolerance test than Formulation A. In conclusion, phospholipidmembranes, that are not perfect or complete spheres, have improvedparticle stability when materials like stearoyl lysophosphatidylcholineor a phenolic compound (such as m-creosol) are added to the membranes.These formulations provide more desirable stability and prevent particleaggregations as compared to formulations lacking such stabilizingcomponents.

Example 3

FIGS. 3-4 illustrate increased chemical stability and particle stabilityobserved upon addition of increasing amounts of lysolecithin into theHDV compositions of the invention.

Addition of lysolecithin, replacing for example a portion of distearoyllecithin (DSPC), prevents a tendency of the HDV to form white flakesduring the first week post manufacture. Without wishing to be limited byany theory, flaking can be caused by fragmented edges of the HDVstructure, which permits HDV units to adhere to each other. In certainembodiments, flaking can be an issue in the manufacturing process,requiring additional filtration steps.

Further, as demonstrated in FIG. 4, lysolecithin formation from DSPC isinhibited by initial addition of lysolecithin to the composition.Further, this occurs without measureable increases in stearic acid.

Taken together, the data presented demonstrates that use of lysolecithinto replace of a portion of DSPC in the compositions of the inventionprovides is advantageous for production of HDV compositions, at leastbecause it allows for a more reliable manufacturing process, reducesoverall number of process steps, and also improves stability of thecompositions of the invention. The disclosures of each and every patent,patent application, and publication cited herein are hereby incorporatedherein by reference in their entirety. While this invention has beendisclosed with reference to specific embodiments, it is apparent thatother embodiments and variations of this invention may be devised byothers skilled in the art without departing from the true spirit andscope of the invention. The appended claims are intended to be construedto include all such embodiments and equivalent variations.

What is claimed is:
 1. A composition comprising a lipid-basednanoparticle, wherein the nanoparticle is enclosed by a bipolar lipidmembrane, which comprises cholesterol, dicetyl phosphate,1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl phosphate (biotin DHPE); wherein the membrane further comprises atleast one agent selected from the group consisting of stearoyllysophosphatidylcholine and m-cresol; wherein the membrane comprisescholesterol, dicetyl phosphate, DSPC, stearoyl lysophosphatidylcholine,m-cresol, and biotin DHPE in a % (w/w) ratio selected from the groupconsisting of: (a) about 9.4:18.1:56.8:14.1:0.0:1.5, (b) about7.7:15.0:58.6:0.0:17.4:1.3, and (c) about 8.4:16.2:47.5:7.6:19.0:1.3;wherein the biotin-DHPE extends outward from the nanoparticle; andwherein the size of the nanoparticle ranges from about 10 nm to about150 nm.
 2. The composition of claim 1, wherein a therapeutic agent isdispersed within the nanoparticle.
 3. The composition of claim 2,wherein the therapeutic agent is covalently bound to the nanoparticle orwherein the therapeutic agent is not covalently bound to thenanoparticle.
 4. The composition of claim 2, wherein the therapeuticagent comprises at least one selected from the group consisting ofinsulin, insulin analogs, interferon, parathyroid hormone, calcitonin,serotonin, serotonin agonist, serotonin reuptake inhibitor, human growthhormone, GIP, anti-GIP monoclonal antibody, metformin, bromocriptine,dopamine, glucagon, amylin and GLP-1.
 5. The composition of claim 2,wherein the nanoparticle is suspended in an aqueous solution comprisinga free dissolved therapeutic agent that is not dispersed within thenanoparticle.
 6. The composition of claim 2, wherein the therapeuticagent is insulin.
 7. The composition of claim 6, wherein thenanoparticle-dispersed insulin and the free dissolved insulin areindependently selected from the group consisting of insulin lispro,insulin aspart, regular insulin, insulin glargine, insulin zinc,extended human insulin zinc suspension, isophane insulin, human bufferedregular insulin, insulin glulisine, recombinant human regular insulin,recombinant human insulin isophane, and any combinations thereof.
 8. Thecomposition of claim 2, further comprising at least one of thefollowing: cellulose acetate phthalate, which is at least partiallybound to the therapeutic agent dispersed within the nanoparticle; atleast one charged organic molecule associated with the therapeutic agentdispersed within the nanoparticle, wherein the charged organic moleculeis at least one selected from the group consisting of protamines,polylysine, poly (arg(-pro-thr))_(n) in a mole ratio of 1:1:1, poly(DL-Ala-poly-L-lys)_(n) in a mole ratio of 6:1, histones, sugar polymerscomprising a primary amino group, polynucleotides with primary aminogroups, proteins comprising amino acid residues with carboxyl (COO⁻) orsulfhydral (S⁻) functional groups, and acidic polymers.
 9. A method ofpreparing the lipid-based nanoparticle of claim 1, the method comprisingcontacting in an aqueous system cholesterol, dicetyl phosphate, DSPC,biotin-DHPE, and the at least one agent.
 10. The method of claim 9,wherein the at least one agent is m-cresol and is added to the aqueoussystem after the cholesterol, dicetyl phosphate, DSPC, stearoyllysophosphatidylcholine if present, and biotin-DHPE had been contactedin the aqueous system.
 11. The method of claim 10, wherein thenanoparticle comprises a therapeutic agent dispersed therewithin. 12.The method of claim 11, wherein one of the following applies: thetherapeutic agent, cholesterol, dicetyl phosphate, DSPC, at least oneagent, and biotin-DHPE are simultaneously contacted in the aqueoussystem; the nanoparticle is formed in the absence of the therapeuticagent, wherein optionally the nanoparticle is at least partiallyconcentrated, purified or isolated, and wherein the therapeutic agent iscontacted with the nanoparticle, whereby at least a portion of thetherapeutic agent is dispersed within the nanoparticle.
 13. Acomposition comprising a lipid-based nanoparticle, wherein thenanoparticle is enclosed by a bipolar lipid membrane comprisingcholesterol, dicetyl phosphate, an amphipathic lipid and a hepatocytereceptor binding molecule; wherein the amphipathic lipid comprises atleast one selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycerol-[3-phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate, and1,2-dipalmitoyl-sn-glycero-3-phosphocholine; wherein the membranecomprises at least one agent selected from the group consisting of astabilizer and stearoyl lysophosphatidylcholine; wherein the stabilizeris selected from the group consisting of m-cresol, benzyl alcohol,methyl 4-hydroxybenzoate, thiomersal, and butylated hydroxytoluene(2,6-di-tert-butyl-4-methylphenol), and wherein, if present, thestabilizer ranges from about 10% to about 25% (w/w) in the membrane;wherein, if present, the stearoyl lysophosphatidylcholine ranges fromabout 5% to about 30% (w/w) in the membrane; wherein the at least onehepatocyte receptor binding molecule extends outward from thenanoparticle; and wherein the size of the nanoparticle ranges from about10 nm to about 150 nm.
 14. The composition of claim 13, wherein atherapeutic agent is dispersed within the nanoparticle.
 15. Thecomposition of claim 14, wherein the therapeutic agent is covalentlybound to the nanoparticle or wherein the therapeutic agent is notcovalently bound to the nanoparticle.
 16. The composition of claim 14,wherein the therapeutic agent comprises at least one selected from thegroup consisting of insulin, interferon, parathyroid hormone,calcitonin, serotonin, serotonin agonist, serotonin reuptake inhibitor,human growth hormone, GIP, anti-GIP monoclonal antibody, metformin,bromocriptine, dopamine, glucagon and GLP-1.
 17. The composition ofclaim 14, wherein the nanoparticle is suspended in an aqueous solutioncomprising a free dissolved therapeutic agent that is not dispersedwithin the nanoparticle.
 18. The composition of claim 14, wherein thetherapeutic agent is insulin.
 19. The composition of claim 18, whereinthe nanoparticle-dispersed insulin and the free dissolved insulin areindependently selected from the group consisting of insulin lispro,insulin aspart, regular insulin, insulin glargine, insulin zinc,extended human insulin zinc suspension, isophane insulin, human bufferedregular insulin, insulin glulisine, recombinant human regular insulin,recombinant human insulin isophane, and any combinations thereof. 20.The composition of claim 13, wherein the amphipathic lipid comprises atleast one selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)],1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl).
 21. Thecomposition of claim 13, wherein the hepatocyte receptor bindingmolecule comprises biotin.
 22. The composition of claim 21, wherein thebiotin-containing hepatocyte receptor binding molecule comprises atleast one selected from the group consisting of N-hydroxysuccinimide(NHS) biotin; sulfo-NHS-biotin; N-hydroxysuccinimide long chain biotin;sulfo-N-hydroxysuccinimide long chain biotin; D-biotin; biocytin;sulfo-N-hydroxysuccinimide-S—S-biotin; biotin-BMCC; biotin-HPDP;iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytinhydrazide; biotin cadaverine; carboxybiotin; photobiotin; ρ-aminobenzoylbiocytin trifluoroacetate; ρ-diazobenzoyl biocytin; biotin DHPE(2,3-diacetoxypropyl2-(5-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethylphosphate); biotin-X-DHPE (2,3-diacetoxypropyl2-(6-(5-(((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)ethyl phosphate); 12-((biotinyl)amino)dodecanoic acid;12-((biotinyl)amino)dodecanoic acid succinimidyl ester; S-biotinylhomocysteine; biocytin-X; biocytin x-hydrazide; biotinethylenediamine;biotin-XL; biotin-X-ethylenediamine; biotin-XX hydrazide; biotin-XX-SE;biotin-XX, SSE; biotin-X-cadaverine; α-(t-BOC)biocytin;N-(biotinyl)-N′-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE;biotin-X-hydrazide; norbiotinamine hydrochloride;3-(N-maleimidylpropionyl)biocytin; ARP; biotin-1-sulfoxide; biotinmethyl ester; biotin-maleimide; biotin-poly(ethyleneglycol) amine; (+)biotin 4-amidobenzoic acid sodium salt; Biotin2-N-acetylamino-2-deoxy-β-D-glucopyranoside;Biotin-α-D-N-acetylneuraminide; Biotin-α-L-fucoside; Biotinlacto-N-bioside; Biotin-Lewis-A trisaccharide; Biotin-Lewis-Ytetrasaccharide; Biotin-α-D-mannopyranoside; and biotin6-O-phospho-α-D-mannopyranoside.
 23. The composition of claim 21,wherein the biotin-containing hepatocyte receptor binding moleculecomprises at least one selected from the group consisting of biotin DHPEand biotin-X-DHPE.
 24. The composition of claim 14, further comprisingat least one of the following: cellulose acetate phthalate, which is atleast partially bound to the therapeutic agent dispersed within thenanoparticle; at least one charged organic molecule bound to thetherapeutic agent dispersed within the nanoparticle, wherein the chargedorganic molecule is at least one selected from the group consisting ofprotamines, polylysine, poly (arg(-pro-thr))_(n) in a mole ratio of1:1:1, poly (DL-Ala-poly-L-lys)_(n) in a mole ratio of 6:1, histones,sugar polymers comprising a primary amino group, polynucleotides withprimary amino groups, proteins comprising amino acid residues withcarboxyl (COO⁻) or sulfhydral (S⁻) functional groups, and acidicpolymers.
 25. The composition of claim 13, wherein at least one applies:the cholesterol ranges from about 5% to about 15% (w/w) in the membrane;the dicetyl phosphate ranges from about 10% to about 25% (w/w) in themembrane; the DSPC ranges from about 40% to about 75% (w/w) in themembrane; hepatocyte receptor binding molecule ranges from about 0.5% toabout 4% (w/w) in the membrane; the amount of the stearoyllysophosphatidylcholine in the membrane is about 5%-30% (w/w) of theamount of DSPC in the membrane.
 26. The composition of claim 13, whereinthe membrane comprises one of the following: (a) cholesterol, dicetylphosphate, DSPC, stearoyl lysophosphatidylcholine, m-cresol, and atleast one selected from the group consisting of biotin DHPE andbiotin-X-DHPE; (b) cholesterol, dicetyl phosphate, DSPC, m-cresol, andat least one selected from the group consisting of biotin DHPE andbiotin-X-DHPE; and (c) cholesterol, dicetyl phosphate, DSPC, stearoyllysophosphatidylcholine, and at least one selected from the groupconsisting of biotin DHPE and biotin-X-DHPE.
 27. A method of preparingthe lipid-based nanoparticle of claim 13, the method comprisingcontacting in an aqueous system cholesterol, dicetyl phosphate,amphipathic lipid, hepatocyte receptor binding molecule, and the atleast one agent.
 28. The method of claim 27, wherein the at least oneagent comprises a stabilizer, which is added to the aqueous system afterthe cholesterol, dicetyl phosphate, amphipathic lipid, stearoyllysophosphatidylcholine if present, and hepatocyte receptor bindingmolecule had been contacted in the aqueous system.
 29. The method ofclaim 27, wherein the nanoparticle comprises a therapeutic agentdispersed therewithin.
 30. The method of claim 29, wherein one of thefollowing applies: the therapeutic agent, cholesterol, dicetylphosphate, amphipathic lipid, hepatocyte receptor binding molecule, andthe at least one agent are simultaneously contacted in the aqueoussystem; the nanoparticle is formed in the absence of the therapeuticagent, wherein optionally the nanoparticle is at least partiallyconcentrated, purified or isolated, and wherein the therapeutic agent iscontacted with the nanoparticle, whereby at least a portion of thetherapeutic agent is dispersed within the nanoparticle.